The biochemical reactions that facilitate lysosomal and nuclear functions may be regulated by phosphorylation or dephosphorylation of the membrane proteins or lipid components. In isolated rat liver lysosome membranes and nuclear envelopes, we have identified phosphatidylinositol (PI) kinase (EC 2.7.1.67) which establishes the presence of the polyphosphoinositide (PPI) metabolic pathway on these membranes. In addition, both phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4 5-bisphosphate (TPI) are labeled in nuclear and lysosomal membranes from hepatocytes cultured with 32Pi. The regulation of these pathways will be investigated by examining the properties of PI kinase from each membrane and the DPI kinase from the cytoplasm. PPI specific phospholipase C and A2 activities will be studied to ascertain whether regulation of their activities occurs through mechanisms such as hormonal stimulated phosphorylation/dephosphorylation as mediated via secondary messengers. PPI in the presence or absence of Ca++ may strongly affect the membrane fluidity and therefore the activity of membrane enzymes. In addition, PPI may have direct effect on specific enzymes such as H+ translocating ATPase on lysosomal membranes or NTPase on nuclear envelopes. The effects of PPI on lysosomal proteolysis rates and on membrane lateral diffusion rates as determined by fluorescence redistribution after photobleaching (FRAP) will be measured in model systems. A single lysosomal membrane proteins is phosphorylated by [Gramma-32p]-ATP, in vitro, and we have proposed that it is a protein subunit of an ATPase. We intend to carry out studies with inhibitors and the [Gramma 35S]-ThioATP analog of ATP to provide correlations between phosphorylation of the subunit and ATPase acitivity. Multiple phosphorylated protein bands have been detected by SDS PAGE of nuclear envelopes incubated with [Gramma-32P]-ATP. We propose to characterize the properties of the protein kinase(s) and to determine whether the proteins which are phosphorylated are modulated by various hormonal treatments in isolated hepatocytes. We believe that it is imporatant to assess the role of polyphosphoinositides in both lysosomal and nuclear membrane functions. Failure of this metabolic pathway to function normally may be an underlying cause of cellular membrane defects, such as in drug-induced lysosomal storage disorders, lysosomal associated silicosis, phagocytosis (Chediak-Higashi disease) and in lysosomal changes in atherosclerosis.